Stabilized exendin-4 compounds

ABSTRACT

The present invention discloses compositions comprising a stabilized Exendin-4 (1-39) and related compounds. The invention describes stabilized Exendin-4 agonists that include at least one modified amino acid residue particularly at positions Gln13, Met14, Trp25, or Asn28 of the Exendin-4 (1-39) molecule. Disclosed are preferred modifications of deaminated, hydrolyzed, oxidized, or isomerized reaction products of the specified amino acid residues corresponding to the same positions in the Exendin-4 (1-39) molecule. The invention also relates to methods of making and using the stabilized Exendin compounds, such as for the treatment of diabetes.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. application Ser. No.10/529,858, filed Apr. 26, 2006, which is the national phase ofInternational Application No. PCT/DK2003/000651, filed Oct. 2, 2003,which, in turn, claims the benefit of U.S. Provisional Application No.60/415,626, filed Oct. 2, 2002. Each of these applications is herebyincorporated by reference.

FIELD OF THE INVENTION

The present invention relates to stabilized Exendin-4 compounds andrelated molecules. In one aspect the invention provides stabilizedExendin-4 agonists that include at least one modified amino acid residueparticularly at positions Gln13, Met14, Trp25, or Asn28 of the Exendin-4(1-39) molecule. Preferred modifications are deaminated, hydrolyzed,oxidized, or isomerized reaction products of the specified amino acidresidues corresponding to the same positions in the Exendin-4 (1-39)molecule. Also provided are methods of making and using the stabilizedExendin compounds. The invention has a broad spectrum of uses andprovides Exendin-4 compounds having better stability when compared toprior Exendin-4 compositions.

BACKGROUND OF THE INVENTION

A number of hormones that lower blood glucose levels are released fromthe gastrointestinal mucosa in response to the presence and absorptionof nutrients in the gut. These include gastrin, secretin,glucose-dependent insulinotropic polypeptide (GIP) and glucagon-likepeptide-1 (GLP-1). The most potent substance known is GLP-1 (Orskov,1992, Diabetologia 35:701-711). Glucagon-like peptide 1 (GLP-1) is aproduct of proglucagon, a 180 amino acid peptide (Drucker, 1998,Diabetes 47:159-169). The overall sequence of proglucagon contains the29-amino acid sequence of glucagon, the 36 or 37 amino acid sequence ofGLP-1 and the 34 amino acid sequence of glucagon-like peptide-2 (GLP-2),an intestinotrophic peptide. GLP-1 has a number of functions. It is aphysiological hormone that enhances the effect on insulin secretion innormal humans and is therefore an incretin hormone. In addition, GLP-1also lowers glucagon concentrations, slows gastric emptying, stimulates(pro)insulin biosynthesis, and enhances insulin sensitivity (Nauck,1997, Horm. Metab. Res. 47:1253-1258). The peptide also enhances theability for the β-cells to sense and respond to glucose in subjects withimpaired glucose tolerance (Byrne, 1998, Eur. J. Clin. Invest.28:72-78). The insulinotropic effect of GLP-1 in humans increases therate of glucose disappearance partly because of increased insulin levelsand partly because of enhanced insulin sensitivity (D'Alessio, 1994,Eur. J. Clin. Invest. 28:72-78). This has placed GLP-1 as a promisingagent for treatment for type II diabetes. Active fragments of GLP-1 havebeen found to be GLP-1 (7-36) and GLP-1 (7-37). However, a majorpharmacological problem with native GLP-1 is its short half-life. Inhumans and rats, GLP-1 is rapidly degraded by dipeptidyl peptidase-IV(DPP-IV) into GLP-1 (9-36)amide, acting as an endogenous GLP-1 receptorantagonist. Several strategies circumventing this problem have beenproposed, some using inhibitors of DPP-IV and others DPP-IV resistantanalogues of GLP-1 (7-36)amide.

Exendins, another group of peptides that lower blood glucose levels havesome sequence similarity (53%) to GLP-1[7-36]NH₂ (Goke et al., 1993, J.Biol. Chem. 268:19650-55). The Exendins are found in the venom ofHelodermatidae or beaded lizards. Exendin-3 is present in the venom ofHeloderma horridum, the Mexican beaded lizard and Exendin-4 is presentin the venom of Heloderma suspectum, the Gila monster. Exendin-4 differsfrom Exendin-3 at just positions two and three. The cDNA encoding theExendin-4 precursor protein, a 47 amino acid peptide fused to the aminoterminus of Exendin-4 has been cloned and sequenced (Pohl et al., 1998,J. Biol. Chem. 273:9778-9784 and WO98/35033).

Exendin-4 is a strong GLP-1 receptor agonist on isolated rat insulinomacells (Goke et al., 1993, J. Biol. Chem. 268:19650-55). Exendin-4 givensystemically lowers blood glucose levels by 40% in diabetic db/db mice(WO99/07404). Recently, Grieg et al. (1999, Diabetologia 42:45-50) haveshown a long lasting blood glucose lowering effect of once dailyintraperitoneal injection of Exendin-4 to diabetic ob/ob mice. U.S. Pat.No. 5,424,286 discloses that a considerable portion of the N-terminalsequence is essential in order to preserve insulinotropic activity(Exendin-4(1-31) and Y³¹-Exendin-4(1-31)) whereas an N-terminallytruncated Exendin (Exendin-4(9-39) has inhibitory properties.

The use of Exendin-3, Exendin-4, and Exendin agonists has been proposedfor the treatment of diabetes mellitus, reducing gastric motility anddelaying gastric emptying and the prevention of hyperglycemia (U.S. Pat.No. 5,424,286, WO 98/05351) as well as for the reduction of food intake(WO 98/30231). Ways of obtaining novel compounds by modifying the nativeExendin sequences have been proposed. One way is to attach lipophilicsubstituents to the molecule, e.g. as described in WO 99/43708 whichdiscloses derivatives of Exendin with just one lipophilic substituentattached to the C-terminal amino acid residue.

A major approach has been to devise Exendin analogues characterised byamino acid substitutions and/or C-terminal truncation of the nativeExendin-4 sequence. This approach is represented by the compounds of WO99/07404, WO 99/25727 and WO 99/25728.

WO 99/07404 discloses Exendin agonists having a general formula I thatdefines a peptide sequence of 39 amino acid residues with Gly Thr inpositions 4-5, Ser Lys Gln in positions 11-13, Glu Glu Glu Ala Val ArgLeu (SEQ ID NO:101) in positions 15-21, Leu Lys Asn Gly Gly (SEQ IDNO:102) in positions 26-30, Ser Ser Gly Ala (SEQ ID NO:103) in positions32-35, and wherein the remaining positions may be occupied by wild-typeExendin amino acid residues or may be occupied by specified amino acidsubstitutions. The formula I does not cover any Exendin agonists oranalogues having specific amino acid deletions and/or being conjugatesas described herein, such as the novel compoundsdesPro³⁶-Exendin-4(1-39) (SEQ ID NO:104), Exendin-4(1-39)-K₆ (SEQ IDNO:105), or desPro³⁶-Exendin-4(1-39)-K₆ (SEQ ID NO:1).

WO 99/25727 discloses Exendin agonists having a general formula I thatdefines a peptide sequence of from 28 to 38 amino acid residues with Glyin position 4 and Ala in position 18, and wherein the remainingpositions may be occupied by wild-type Exendin amino acid residues ormay be occupied by specified amino acid substitutions. Formula I doesnot comprise a peptide sequence having Ser as the C-terminal amino acidand Exendin agonists or analogues having specific amino acid deletionsand/or being conjugates as described herein, such as the novel compoundsdesPro³⁶-Exendin-4(1-39) (SEQ ID NO:104), Exendin-4(1-39)-K₆ (SEQ IDNO:105), or desPro³⁶-Exendin-4(1-39)-K₆ (SEQ ID NO:1). Further, formulaII of WO 99/25727 defines a peptide sequence similar to formula I, butincluding Exendin derivatives having a C(1-10)alkanoyl orcycloalkylalkanoyl substituent on lysine in position 27 or 28.

When treating inappropriate post-prandial blood glucose levels thecompounds are administered frequently, for example one, two or threetimes a day.

WO 99/25728 discloses Exendin agonists having a general formula I thatdefines a peptide sequence of from 28 to 39 amino acid residues withfixed Ala in position 18, and wherein the remaining positions may beoccupied by wild-type Exendin amino acid residues or may be occupied byspecified amino acid substitutions. Said Exendin agonists all correspondto a truncated Exendin analogue having a varying degree of amino acidsubstitutions. Peptide sequences of from 34 to 38 amino acid residues donot have Ser C-terminally. A peptide sequence of 39 amino acid residuesmay have either Ser or Tyr C-terminally, but no further residues.Exendin agonists or analogues having specific amino acid deletionsand/or being conjugates according to the invention described herein arenot comprised by formula I. Further, formula II defines a peptidesequence similar to formula I, but including Exendin derivatives havinga C(1-10)alkanoyl or cycloalkylalkanoyl substituent on lysine inposition 27 or 28.

WO 99/46283 (published 16 Sep. 1999) discloses peptide conjugatescomprising a pharmacologically active peptide X and a stabilisingpeptide sequence Z of 4-20 amino acid residues covalently bound to X,where said conjugates are characterised in having an increased half-lifecompared to the half-life of X. X may be Exendin-4 or Exendin-3.

It would be desirable to have Exendin compositions that can providebetter stability than prior compounds. Further desirable would be tohave Exendin compositions that can resist degradation. Such compositionswould be especially useful in settings where significant storage timesare expected and/or where there is risk from unwanted oxidation,hydrolysis or deamination reactions.

SUMMARY OF THE INVENTION

The present invention generally relates to stabilized Exendin-4compounds and related compositions. In one aspect, the inventionprovides stabilized Exendin compounds, particularly Exendin-4 agonists,that include at least one modified amino acid residue at positionsGln13, Met14, Trp25, or Asn28 of the Exendin-4 (1-39) molecule. Alsoprovided are methods of making and using the stabilized Exendin-4compositions. The invention has a broad spectrum of uses includingproviding Exendin-4 compositions with better stability and storageproperties when compared with prior Exendin-4 compositions and relatedcompounds.

We have discovered that it is possible to stabilize Exendin-4 compoundsand related compositions by modifying particular Exendin amino acidstherein. Preferred modifications according to the invention aredeaminated, hydrolyzed, oxidized, or isomerized reaction products of thespecified amino acid residues of the Exendin-4 (1-39) molecule. Suchcompounds can be readily made by one or a combination of standardtechniques including exposing Exendin-4 (1-39) to pharmaceuticallyacceptable formulation procedures, exposing the molecule to potentiallyreactive conditions such as contact with water, oxygen, light, heat, orthe like, or otherwise providing conditions conducive to spontaneous orsemi-spontaneous degradation of amino acids corresponding to at leastone of Gln13, Met14, Trp25, or Asn28 of the Exendin-4 (1-39) molecule.

Practice of preferred embodiments of the invention provide substantialadvantages. For instance, use of the invention is able to providestabilized Exendin-4 compounds that feature more reliable activityparticularly over prolonged storage times. Such stabilized Exendin-4molecules and related compositions can assist medical uses includingclinical studies and other uses by providing more reproducible andconsistent agonist activity. This feature of the invention isparticularly important when multiple Exendin-4 preparations (lots) areneeded. That is, by stabilizing the Exendin-4 compounds and relatedmolecules according to the invention, it is now possible to improveconsistency between lots.

Additionally, use of the invention can now provide more consistentdosing of Exendin-4 compositions and related molecules. That is, thestabilized compounds of the invention are less apt to degrade and thuscan provide more reliable therapeutic activity. This feature of theinvention is particularly important when it is desirable to produceExendin-4 and related molecules having relatively consistent activity orgood stability on a moderate or large scale.

By the phrase “Exendin-4 compound or related molecule” including pluralforms is meant Exendin-4 (1-39), or a variant, analogue or derivativethereof as defined in this application. Illustrative is Exendin-4 (1-39)and derivatives thereof that include a deletion of between from about 1to about 5 amino acids that corresponds to positions 34, 35, 36, 37 or38 of Exendin-4. Optionally, such derivatives further include at leastone peptide Z as defined herein. Such compounds or molecules are“stabilized” according to the invention (eg., a stabilized Exendin-4(1-39) compound) by modifying at least one of the amino acid residues:Gln13, Met14, Trp25, or Asn28 of the Exendin-4 (1-39) molecule.Preferably, one or two of such amino acids are modified as discussedalthough more can be to suit an intended use.

Accordingly, and in one embodiment, the invention provides a compoundthat includes at least one stabilized Exendin-4 (1-39), preferably one,two or three of same, preferably one of such a stabilized Exendin-4(1-39). In one embodiment, the compound includes:

a) a deletion of 0 to 5 amino acids at positions corresponding topositions 34-38 of Exendin-4 (1-39),

b) optionally, at least one peptide sequence Z comprising 4-20 aminoacid units covalently bound to said compound; and at least one of thefollowing:

i) an Asn residue having a deaminated side chain, an Asn residue havinghydrolyzed side chain or a structural isomer of an Asp residue, whereinthe Asn or Asp residue corresponds to position 28 of Exendin-4,

ii) an oxidized methionine residue corresponding to position 14 ofExendin-4,

iii) an oxidized tryptophan residue corresponding to position 25 ofExendin-4,

iv) a deaminated or hydrolyzed Gln corresponding to position 13 ofExendin-4 and a pharmaceutically acceptable salt or solvate thereof.

In one embodiment of the forgoing compound, the stabilized Exendin-4(1-39) includes at least one L-amino acid residue or at least oneD-amino acid residue. Alternatively, the compound includes combinationsof L- and D-amino acid residues.

In another aspect, the present invention provides a compound thatincludes at least one stabilized Exendin-4 (1-39), preferably two, threeor four of same, more preferably one of such stabilized Exendin-4 (1-39)molecules. In one embodiment, the compound includes at least one of:

i) an Asn residue having a deaminated side chain, an Asn residue havinghydrolyzed side chain or a structural isomer of an Asp residue, whereinthe Asn or Asp residue corresponds to position 28 of Exendin-4,

ii) an oxidized methionine residue corresponding to position 14 ofExendin-4,

iii) an oxidized tryptophan residue corresponding to position 25 ofExendin-4,

iv) a deaminated or hydrolyzed Gln corresponding to position 13 ofExendin-4; and a pharmaceutically acceptable salt or solvate thereof.

Further provided are pharmaceutically acceptable compositions thatinclude at least one, preferably two, three, or four of the stabilizedExendin-4 (1-39) molecules, more preferably one of such compounds.Examples of such compositions are provided below.

In another aspect, the invention provides a method of making astabilized Exendin-4 composition or related molecule as disclosedherein. In one embodiment, the method includes at least one of thefollowing steps:

a) obtaining Exendin-4 (1-39) or a variant, analogue, or derivativethereof; and

b) incubating the Exendin-4 (1-39) or the variant, analogue, orderivative thereof under conditions sufficient to introduce at least oneof the following amino acids therein:

i) an Asn residue having a deaminated side chain, an Asn residue havinghydrolyzed side chain or a structural isomer of an Asp residue, whereinthe Asn or Asp residue corresponds to position 28 of Exendin-4,

ii) an oxidized methionine residue corresponding to position 14 ofExendin-4,

iii) an oxidized tryptophan residue corresponding to position 25 ofExendin-4

iv) a deaminated or hydrolyzed Gln corresponding to position 13 ofExendin-4; and a pharmaceutically acceptable salt or solvate thereof.

It will often be preferred to make the stabilized Exendin-4 molecules ofthe invention synthetically or semi-synthetically. An example of such amethod is described below and includes use of the Merrifield peptidesynthesis technique.

In particular embodiments the foregoing methods further include the stepof detecting presence or absence of at least one of amino acids(i)-(iv). Alternatively, or in addition, the method can includeidentifying at least one of the amino acids (i)-(iv) in the composition.Methods for detecting and identifying the modified amino acids arediscussed below.

In another embodiment, the invention features a method of making thestabilized Exendin-4 compound and related molecules disclosed herein.Typical of such methods includes at least one of the following steps:

a) obtaining Exendin-4 (1-39) or a variant, analogue or derivativethereof,

b) contacting the Exendin-4 (1-39) or the variant, analogue orderivative with at least one pharmaceutically acceptable carrier orvehicle to produce a mixture; and

c) incubating the mixture under conditions sufficient to introduce atleast one of the following amino acids therein:

i) an Asn residue having a deaminated side chain, an Asn residue havinghydrolyzed side chain or a structural isomer of an Asp residue, whereinthe Asn or Asp residue corresponds to position 28 of Exendin-4,

ii) an oxidized methionine residue corresponding to position 14 ofExendin-4,

iii) an oxidized tryptophan residue corresponding to position 25 ofExendin-4 iv) a deaminated or hydrolyzed Gln corresponding to position13 of Exendin-4; and a pharmaceutically acceptable salt or solvatethereof.

Such a method can also further include the step of detecting presence orabsence of at least one of the amino acid residues (i)-(iv).Alternatively, or in addition, the method can include identifying atleast one of the amino acids (i)-(iv) in the composition.

In another aspect, the invention provides a method of stabilizingExendin-4 (1-39) or a variant, analogue or derivative thereof. In oneembodiment, the method includes at least one of the following steps:

a) obtaining Exendin-4 (1-39) or a variant, derivative or analoguethereof; and

b) incubating the Exendin-4 (1-39) or the variant, derivative oranalogue under conditions sufficient to introduce at least one of thefollowing amino acid residues therein:

i) an Asn residue having a deaminated side chain, an Asn residue havinghydrolyzed side chain or a structural isomer of an Asp residue, whereinthe Asn or Asp residue corresponds to position 28 of Exendin-4,

ii) an oxidized methionine residue corresponding to position 14 ofExendin-4,

iii) an oxidized tryptophan residue corresponding to position 25 ofExendin-4; and

iv) a deaminated or hydrolyzed Gln corresponding to position 13 ofExendin-4; and a pharmaceutically acceptable salt or solvate thereof.

In another aspect, the invention provides a method for treating diabetestype 1 or type 2, insulin resistance syndrome, impaired glucosetolerance (IGT), obesity, eating disorders, hyperglycemia, metabolicdisorders, and gastric disease. In one embodiment, the method includesadministering a therapeutically effective amount of at least one of thestabilized Exendin-4 compounds or related molecules disclosed herein.

The invention also provides a method for treating disease statesassociated with elevated blood glucose levels. In one embodiment, themethod includes administering a therapeutically effective amount of atleast one of the stabilized Exendin-4 compounds or related moleculesdisclosed herein.

Also provided is a method for regulation of blood glucose levels. In oneembodiment, the method includes administering a therapeuticallyeffective amount of at least one of at least one of the stabilizedExendin-4 compounds or related molecules disclosed herein.

The invention also provides a method for regulation of gastric emptying.In one embodiment, the method includes administering a therapeuticallyeffective amount of at least one of the stabilized Exendin-4 compoundsor related molecules provided herein.

The present invention also provides a method of stimulating insulinrelease in a mammal. In one embodiment, the method includesadministering an effective insulinotropic amount of at least one of thestabilized Exendin-4 compounds disclosed herein.

Additionally provided is a method of lowering blood glucose level in amammal. In one embodiment, the method includes administering an amountof at least one of the stabilized Exendin-4 compounds or relatedmolecules described herein in an amount effective to lower blood glucoselevel in said mammal.

The invention also provides a method of lowering plasma lipid level in amammal. In one example, the method includes administering an amount ofat least one of the stabilized Exendin-4 compounds described herein inan amount effective to lower plasma lipid level in said mammal.

Also provided is a method of reducing mortality and morbidity aftermyocardial infarction in a mammal. In one embodiment, the methodincludes administering an amount of at least one of the stabilizedExendin-4 compounds disclosed herein in an amount effective to reducemortality and morbidity after myocardial infarction.

Also provided is a method of stimulating insulin release in a mammal. Inone embodiment, the method includes administering an effectiveinsulinotropic amount of at least one of the stabilized Exendin-4compounds provided herein.

Preferably, the mammal featured in each of the foregoing methods is aprimate, preferably a human patient in need of treatment.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the effect of Compound 1 on glucose tolerance in mice.

FIG. 2 shows the effect of Compound 14 on glucose tolerance in mice.

FIG. 3 shows the effect of Compound 6 on glucose tolerance in mice.

FIG. 4 shows the effect of Compound 7 on glucose tolerance in mice.

FIG. 5 shows the effect of Compound 1 on glucose tolerance in mice.

FIG. 6 shows the effect of Compound 5 on glucose tolerance in mice.

FIG. 7 shows the effect of Compound 2 on glucose tolerance in mice.

FIG. 8 depicts the influence of Compounds 1, 8, 9, 10, and 11 on bloodglucose levels in mice.

FIG. 9 shows the sequences of Compound 1 (des Pro³⁶Exendin-4 (1-39)-K₆)and stabilized compounds of Compound 1 (SEQ ID NO:1), namely Compound 2(SEQ ID NO:2), 3 (SEQ ID NO:3), 4 (SEQ ID NO:4), 5 (SEQ ID NO:5), 6 (SEQID NO:6), 7 (SEQ ID NO:7), 11 (SEQ ID NO:11), 12 (SEQ ID NO:12), 13 (SEQID NO:13), and 14 (SEQ ID NO:14), as well as stabilized compounds ofExendin-4 (1-39), namely Compounds 8 (SEQ ID NO:8), 9 (SEQ ID NO:9), and10 (SEQ ID NO:10).

DETAILED DESCRIPTION OF THE INVENTION

As discussed, the invention provides stabilized Exendin agonists thatinclude at least one modified amino acid residue particularly atpositions Gln13, Met14, Trp25, or Asn28 of the Exendin-4 (1-39)molecule. Preferred modifications are deaminated, hydrolyzed, oxidized,or isomerized reaction products of the specified amino acid residues ofthe Exendin-4 (1-39) molecule. Also provided are methods of making andusing the stabilized Exendin compounds.

Preferred stabilized Exendin-4 (1-39) compounds and pharmaceuticallyacceptable salt thereof which include:

a) a deletion of 0 to 5 amino acids at positions corresponding topositions 34-38 of Exendin-4,

b) optionally, at least one peptide sequence Z comprising 4-20 aminoacid units covalently bound to said variant; and at least one of thefollowing:

-   -   i) an Asn residue having a deaminated side chain, an Asn residue        having hydrolyzed side chain or a structural isomer of an Asp        residue, wherein the Asn or Asp residue corresponds to position        28 of Exendin-4,    -   ii) an oxidized methionine residue corresponding to position 14        of Exendin-4,    -   iii) an oxidized tryptophan residue corresponding to position 25        of Exendin,    -   iv) a deaminated or hydrolyzed Gln corresponding to position 13        of Exendin-4; and a pharmaceutically acceptable salt or solvate        thereof.

In one embodiment, the Asn residue is an α-aspartate (Asp) orβ-aspartate (isoaspartyl) residue or the Asn residue is an Cyclic imide.In another embodiment the oxidized methionine residue is a methioninylsulfoxide or a methioninyl sulfone. In embodiments in which the Cyclicimide is derived from an Asp or Gln residue, the cyclyzed product issometimes referred to herein as an aspartimide or glutimide,respectively.

Alternatively, or in addition, the oxidized tryptophan residue includesan oxidized 3H-indol-3-yl group. Other examples include an oxidizedtryptophan residue that is N-formylkynurenine (NFK), 3-hydroxykynurenine(3-OH-KYN), hydroxytryptophan (HTRP), or kynurenine (KYN).

In embodiments in which the stabilized Exendin-4 compound or relatedcomposition includes at least one Z peptide and preferably one or two ofsame, Z comprises at least one Lys amino acid unit, typically betweenabout 4 to about 20 Lys amino acid units, preferably about 6 Lys (SEQ IDNO:106) amino acid units.

In embodiments in which the stabilized Exendin-4 compound includes a Zpeptide, that peptide is covalently bound by a peptide bond. Forinstance, Z is covalently bound to the stabilized Exendin-4 (1-39)compound at the C-terminal carbonyl function.

More particular stabilized Exendin-4 (1-39) compounds according to theinvention are represented by the following sequences:

des Pro³⁶[Asp²⁸]Exendin-4 (1-39) (SEQ ID NO:15),

des Pro³⁶[IsoAsp²⁸]Exendin-4 (1-39) (SEQ ID NO:16),

des Pro³⁶[Cyclic imide²⁸]Exendin-4 (1-39) (SEQ ID NO:17),

des Pro³⁶[Met(O)¹⁴]Exendin-4 (1-39) (SEQ ID NO:18),

des Pro³⁶[Trp(O₂)²⁵]Exendin-4 (1-39) (SEQ ID NO:19),

des Pro³⁶[Met(O)¹⁴, Asp²⁸]Exendin-4 (1-39) (SEQ ID NO:20),

des pro³⁶[Met(O)¹⁴, IsoAsp²⁸]Exendin-4 (1-39) (SEQ ID NO:21),

des Pro³⁶[Met(O)¹⁴, Cyclic imide²⁸]Exendin-4 (1-39) (SEQ ID NO:22),

des Pro³⁶[Met(O)¹⁴, Trp(O₂)²⁵]Exendin-4 (1-39) (SEQ ID NO:23),

des pro³⁶[Met(O)¹⁴ Trp(O₂)²⁵, Asp²⁸]Exendin-4 (1-39) (SEQ ID NO:24),

des Pro³⁶[Met(O)¹⁴ Trp(O₂)²⁵, IsoAsp²⁸]Exendin-4 (1-39) (SEQ ID NO:25),

des Pro³⁶[Met(O)¹⁴ Trp(O₂)²⁵, Cyclic imide²⁸]Exendin-4 (1-39) (SEQ IDNO:26), or

a pharmaceutically acceptable salt or solvate thereof.

With respect to any of the forgoing stabilized Exendin-4 (1-39)compounds, the compounds may further include a deaminated or hydrolyzedGln corresponding to position 13 of Exendin-4.

In one embodiment of the forgoing compounds, each sequence is attachedat the N- or C-terminus to the following group: -Lys₆-NH₂ (SEQ IDNO:106). Preferably, the group is attached to the C-terminus of thesequence.

Additionally specific stabilized Exendin-4 (1-39) compounds include thefollowing sequences:

H-(Lys)₆-des Pro³⁶[Asp²⁸]Exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO:27) desAsp²⁸ Pro³⁶, Pro³⁷, Pro³⁸Exendin-4(1-39)-NH₂ (SEQ ID NO:28),H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸[Asp²⁸]Exendin-4(1-39)-NH₂ (SEQ IDNO:29) H-Asn-(Glu)₅ des Pro , Pro³⁷, Pro³⁸[Asp²⁸]Exendin-4(1-39)-NH₂(SEQ ID NO:30), des Pro³⁶, Pro³⁷, Pro³⁸[Asp²⁸]Exendin-4(1-39)-(Lys)₆-NH₂(SEQ ID NO:31), H-(Lys)₆-des Pro³⁶, Pro³⁷,Pro³⁸[Asp²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:32), H-Asn-(Glu)₅-desPro³⁶, Pro³⁷, Pro³⁸[Asp²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:33),

H-(Lys)₆-des Pro³⁶[Cyclic imide²⁸]Exendin-4(1-39)-Lys₆-NH₂ (SEQ IDNO:34),des Pro³⁶, Pro³⁷, Pro³⁸[Cyclic imide²⁸]Exendin-4(1-39)-NH₂ (SEQ IDNO:35),H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸[Cyclic imide²⁸]Exendin-4(1-39)-NH² (SEQID NO:36)H-Asn-(Glu)₅-des Pro³⁶, Pro³⁷, Pro³⁸[Cyclicimide²⁸]Exendin-4(1-39)-NH²(SEQ ID NO:37),des Pro³⁶, Pro³⁷, Pro³⁸[Cyclic imide²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQID NO:38),H-(Lys)₆ des Pro³⁶, Pro³⁷, Pro³⁸[Cyclicimide²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:39),H-Asn-(Glu)₅-des Pro³⁶, Pro³⁷, Pro³⁸[Cyclicimide²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:40),

H-(Lys)₆-des Pro³⁶[Trp(O₂)²⁵, Asp²⁸]Exendin-4(1-39)-Lys₆-NH₂ (SEQ IDNO:41), H-des Asp²⁸ Pro³⁶, Pro³⁷, Pro³⁸[Trp(O₂)²⁵]Exendin-4(1-39)-NH₂(SEQ ID NO:42), H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸[Trp(O₂)²⁵,Asp²⁸]Exendin-4(1-39)-NH₂ (SEQ ID NO:43), H-Asn-(Glu)₅-des Pro³⁶, Pro³⁷,Pro³⁸[Trp(O₂)²⁵, Asp²⁸]Exendin-4(1-39)-NH₂ (SEQ ID NO:44), des Pro³⁶,Pro³⁷, Pro³⁸[Trp(O₂)²⁵, Asp²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:45),

H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸[Trp(O₂)²⁵,Asp²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:46),H-Asn-(Glu)₅-des Pro³⁶, Pro³⁷, Pro³⁸[Trp(O₂)²⁵,Asp²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:47),H-(Lys)₆-des Pro³⁶[Trp(O₂)²⁵, Cyclic imide²⁸]Exendin-4(1-39)-Lys₆-NH₂(SEQ ID NO:48),des Cyclic imide²⁸ Pro³⁶, Pro³⁷, Pro³⁸[Trp(O₂)²⁵]Exendin-4(1-39)-NH₂(SEQ ID NO:49),H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸[Trp(O₂)²⁵, Cyclicimide²⁸]Exendin-4(1-39)-NH₂ (SEQ ID NO:50),H-Asn-(Glu)₅-des Pro³⁶, Pro³⁷, Pro³⁸[Tip(O₂)₂₅, Cyclicimide²⁸]Exendin-4(1-39)-NH₂ (SEQ ID NO:51),des Pro³⁶, Pro³⁷, Pro³⁸[Trp(O₂)²⁵, Cyclicimide²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:52),H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸[Trp(O₂)²⁵, Cyclicimide²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:53),H-Asn-(Glu)₅-des Pro³⁶, Pro³⁷, Pro³⁸[Trp(O₂)²⁵, Cyclicimide²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:54),

H-(Lys)₆-des Pro³⁶[Met(O)₄, Asp²⁸]Exendin-4(1-39)-Lys₆-NH₂ (SEQ IDNO:55), des Met(O)¹⁴Asp²⁸ Pro³⁶, Pro³⁷, Pro³⁸ Exendin-4(1-39)-NH₂ (SEQID NO:56), H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸[Met(O)¹⁴,Asp²⁸]Exendin-4(1-39)-NH₂ (SEQ ID NO:57), H-Asn-(Glu)₅-des Pro³⁶, Pro³⁷,Pro³⁸[Met(O)¹⁴, Asp²⁸]Exendin-4(1-39)-NH₂ (SEQ ID NO:58),

des Pro³⁶, Pro³⁷, pro³⁸[Met(O)¹⁴, Asp²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQID NO:59),H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸[Met(O)¹⁴,Asp²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:60),H-Asn-(Glu)₅ des Pro³⁶, Pro³⁷, Pro³⁸[Met(O)¹⁴,Asp²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:61),H-Lys₆-des Pro³⁶[Met(O)¹⁴, Cyclic imide²⁸]Exendin-4(1-39)-Lys₆-NH₂ (SEQID NO:62),des Pro³⁶, Pro³⁷, Pro³⁸[Met(O)¹⁴, Cyclic imide²⁸]Exendin-4(1-39)-NH₂(SEQ ID NO:63),H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸[Met(O)¹⁴, Cyclicimide²⁸]Exendin-4(1-39)-NH₂ (SEQ ID NO:64),H-Asn-(Glu)₅-des Pro³⁶, Pro³⁷, Pro³¹[Met(O)¹⁴, Cyclicimide²⁸]Exendin-4(1-39)-NH₂ (SEQ ID NO:65),des Pro³⁶, Pro³⁷, Pro³⁸[Met(O)¹⁴, Cyclicimide²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:66),H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸[Met(O)¹⁴, Cyclicimide²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:67),H-Asn-(Glu)₅-des Pro³⁶, Pro³⁷, Pro³⁸[Met(O)¹⁴, Cyclicimide²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:68),

H-Lys₆-des Pro³⁶[Met(O) 1⁴,Trp(O₂)²⁵,Asp²⁸]Exendin-4(1-39)-Lys₆-NH₂ (SEQID NO:69), H-des Asp²⁸ Pro³⁶, Pro³⁷,Pro³⁸[Met(O)¹⁴,Trp(O₂)²⁵]Exendin-4(1-39)-NH₂ (SEQ ID NO:70),

H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸[Met(O)¹⁴, Trp(O₂)²⁵,Asp²⁸]Exendin-4(1-39)-NH₂ (SEQ ID NO:71),H-Asn-(Glu)₅-des Pro³⁶, Pro³⁷, Pro³⁸[Met(O)¹⁴, Trp(O₂)²⁵,Asp²⁸]Exendin-4(1-39)-NH₂ (SEQ ID NO:72),

des Pro , Pro³⁷, Pro³⁸[Met(O)¹⁴, Trp(O₂)²⁵,Asp²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:73),

H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸[Met(O)¹⁴, Trp(O₂)²⁵,Asp²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:74),H-Asn-(Glu)₅-des Pro³⁶, Pro³⁷, Pro³⁸[Met(O)¹⁴, Trp(O₂)²⁵,Asp²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:75),H-Lys₆-des Pro³⁶[Met(O)¹⁴, Trp(O₂)²⁵, Cyclicimide²⁸]Exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO:76),des Cyclic imide²⁸ Pro³⁶, Pro³⁷, Pro³⁸[Met(O)¹⁴,Trp(O₂)²⁵]Exendin-4(1-39)-NH₂ (SEQ ID NO:77),H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸[Met(O)¹⁴, Trp(O₂)²⁵, Cyclicimide²⁸]Exendin-4(1-39)-NH₂ (SEQ ID NO:78),H-Asn-(Glu)₅-des Pro³⁶, Pro³⁷, Pro³⁸[Met(O)¹⁴, Trp(O₂)²⁵, Cyclicimide²⁸]Exendin-4(1-39)-NH₂ (SEQ ID NO:79),des Pro³⁶, Pro³⁷, Pro³⁸[Met(O)¹⁴, Trp(O₂)²⁵, Cyclicimide²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:80),H-(Lys)₆-des Pro³⁶, Pro³⁷, Pro³⁸[Met(O)¹⁴, Trp(O₂)²⁵, Cyclicimide²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:81),H-Asn-(Glu)₅-des Pro³⁶, Pro³⁷, Pro³⁸[Met(O)¹⁴, Trp(O₂)²⁵, Cyclicimide²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:82),or a pharmaceutically acceptable salt or solvate thereof.

With respect to any of the foregoing stabilized Exendin-4 (1-39)compounds, the compounds may further include a deaminated or hydrolyzedGln corresponding to position 13 of Exendin-4.

As also discussed, the invention features stabilized Exendin-4(1-39)compounds that include at least one of:

i) an Asn residue having a deaminated side chain, an Asn residue havinghydrolyzed side chain or a structural isomer of an Asp residue, whereinthe Asn or Asp residue corresponds to position 28 of Exendin-4,

ii) an oxidized methionine residue corresponding to position 14 ofExendin-4,

iii) an oxidized tryptophan residue corresponding to position 25 ofExendin-4; and

iv) a deaminated or hydrolyzed Gln corresponding to position 13 ofExendin-4;

and a pharmaceutically acceptable salt or solvate thereof.

Salts and Solvates

Also envisioned are pharmaceutically acceptable salts or solvates ofsuch compounds. Examples of such stabilized Exendin-4(1-39) compoundsinclude the following sequences:

[Asp²⁸]Exendin-4 (1-39) (SEQ ID NO:83),

[IsoAsp²⁸]Exendin-4 (1-39) (SEQ ID NO:84),

[Cyclic imide²⁸]Exendin-4 (1-39) (SEQ ID NO:85),

[Met(O)¹⁴]Exendin-4 (1-39) (SEQ ID NO:86),

[Trp(O₂)²⁵]Exendin-4 (1-39) (SEQ ID NO:87),

[Met(O)¹⁴, Asp²⁸]Exendin-4 (1-39) (SEQ ID NO:88),

[Met(O)¹⁴, IsoAsp²⁸]Exendin-4 (1-39) (SEQ ID NO:89),

[Met(O)¹⁴, Cyclic imide²⁸]Exendin-4 (1-39) (SEQ ID NO:90),

[Met(O)¹⁴, Trp(O₂)²⁵]Exendin-4 (1-39) (SEQ ID NO:91),

[Trp(O₂)²⁵, Asp²⁸]Exendin-4 (1-39) (SEQ ID NO:92),

[Trp(O₂)²⁵, IsoAsp²⁸]Exendin-4 (1-39) (SEQ ID NO:93),

[Trp(O₂)²⁵, Cyclic imide²⁸]Exendin-4 (1-39) (SEQ ID NO:94),

[Met(O)¹⁴, Trp(O₂)₂₅, ASP28]Exendin-4 (1-39) (SEQ ID NO:95)

[Met(O)¹⁴, Trp(O₂)²⁵, IsoAsp28]Exendin-4 (1-39) (SEQ ID NO:96), or

[Met(O)¹⁴, Trp(O₂)²⁵, Cyclic imide²⁸]Exendin-4 (1-39) (SEQ ID NO:97).

and a pharmaceutically acceptable salt or solvate thereof.

With respect to any of the forgoing stabilized Exendin-4 (1-39)compounds, the compounds may further include a deaminated or hydrolyzedGln corresponding to position 13 of Exendin-4.

L and D Variants

The stabilized Exendin-4 (1-39) compounds disclosed herein includingvariants, analogue and derivatives thereof, can feature at least oneamino acid in the L- or D-configuration (or both D- and L-forms). Inembodiments in which at least one of the amino acid residues isdeaminated, the stabilized Exendin-4 (1-39) compound can have at leastone amino acid residue in the L-form, at least one amino acid in theD-form, or combinations thereof.

Additionally preferred stabilized Exendin-4 (1-39) compounds and relatedmolecules of the invention feature good biological activity. Morepreferred are those compounds that exhibit at least 70% of thebiological activity of the corresponding unstabilized Exendin-4 (1-39)compound or related molecule, more preferably at least 80%, 90% orgreater activity up to about 100% of that biological activity. Methodsfor testing the biological activity of a variety of Exendin-4 (1-39)compounds and related molecules have been disclosed WO 01/04156(hereinafter “PCT/DK00/00393”), EP application 99610043.4 and U.S.provisional application 60/143,591; the disclosures of which areincorporated herein by reference.

For example, one acceptable test for Exendin-4 (1-39) biologicalactivity is the blood glucose assay in diabetic ob/ob mice described inthe PCT/DK00/00393 application.

It is an object of the invention to provide a pharmaceuticallyacceptable composition that includes at least one of the stabilizedExendin-4 (1-39) compounds or related molecules disclosed herein.Preferably, such a composition will include less than about 5 compounds,such as two identical compounds.

A wide variety of pharmaceutically acceptable formulations are known inthe field and have been disclosed in the PCT/DK00/00393, EP application99610043.4 and U.S. provisional application 60/143,591.

Formulation

For instance, and as provided in the PCT/DK00/00393 case, an Exendin-4(1-39) compound or related molecule in accord with the invention can becombined with one or more physiologically acceptable carriers. Suchcompositions may be in a form adapted to oral, parenteral (includingsubcutaneous (s.c.), intravenous (i.v.), intramuscular (i.m.), epidural,direct brain and intraperitoneal (i.p.)), rectal, intratracheal,intranasal, dermal, vaginal, buccal, ocularly, or pulmonaryadministration, preferably in a form adapted to subcutaneous or oraladministration, and such compositions may be prepared in a mannerwell-known to the field. See generally described in “Remington'sPharmaceutical Sciences”, 17. Ed. Alfonso R. Gennaro (Ed.), MarkPublishing Company, Easton, Pa., U.S.A., 1985 and more recent editionsand in the monographs in the “Drugs and the Pharmaceutical Sciences”series, Marcel Dekker. The compositions may appear in conventionalforms, for example, capsules, tablets, aerosols, topical applicationforms, liquid or semiliquid forms, such as solutions, suspensions,dispersions, emulsions, micelles or liposomes. Preferred are liquidcompositions suitable for s.c. administration. In a preferredembodiment, the compositions of the present invention are administeredsubcutaneously. In an alternative preferred embodiment, the compositionsof the present invention are administered orally, and in such cases onepreferred administration form is a tablet or capsule.

The pharmaceutical carrier or diluent employed may be a conventionalsolid or liquid carrier. Examples of solid carriers are lactose, terraalba, sucrose, cyclodextrin, talc, gelatin, agar, pectin, acacia,magnesium stearate, stearic acid or lower alkyl ethers of cellulose.Examples of liquid carriers are syrup, peanut oil, olive oil,phospholipids, sterols, fatty acids, fatty acid amines, polyoxyethylene,isotonic buffer solutions and water. Similarly, the carrier or diluentmay include any sustained release material known in the art, such asglyceryl monostearate or glyceryl distearate, alone or mixed with a wax.If a solid carrier is used for oral administration, the preparation maybe in the form of a tablet, placed in a hard gelatin capsule in powderor pellet form or it can be in the form of a troche or lozenge. Theamount of solid carrier will vary widely but will usually be from about25 mg to about 1 g.

A typical tablet, which may be prepared by conventional tablettingtechniques may contain:

Core: active compound (as free compound of the invention or saltthereof) 100 mg; colloidal silicon dioxide (Aerosil) 1.5 mg; cellulose,microcryst. (Avicel) 70 mg; modified cellulose gum (Ac-Di-Sol) 7.5 mg;magnesium stearate.

Coating: HPMC approx. 9 mg; *Mywacett 9-40T approx. 0.9 mg; *acylatedmonoglyceride used as plasticizer for film coating.

If a liquid carrier is used, the preparation may be in the form of asyrup, emulsion, soft gelatin capsule, or sterile injectable liquid suchas an aqueous or non-aqueous liquid suspension or solution.

For nasal administration, the preparation may contain a compound of thepresent invention, preferably a conjugate, dissolved or suspended in aliquid carrier, in particular, an aqueous carrier, for aerosolapplication. The carrier may contain additives such as solubilizingagents, e.g., propylene glycol, surfactants such as bile acid salts orpolyoxyethylene higher alcohol ethers, absorption enhancers such aslecithin (phosphatidylcholine) or cyclodextrin, or preservatives such asparabines.

A stabilized Exendin-4 (1-39) compound or related molecule of theinvention may also be in a form suited for local or systemic injectionor infusion and may, as such, be formulated with sterile water or anisotonic saline or glucose solution. The compositions may be sterilizedby conventional sterilization techniques, which are well known in theart. The resulting aqueous solutions may be packaged for use or filteredunder aseptic conditions and lyophilized, the lyophilized preparationbeing combined with the sterile aqueous solution prior toadministration. Preferably, the formulation to be used for intravenous,subcutaneous and oral dosing will be a solution of the active compoundin buffer. The preparation may be produced immediately before use fromactive drug substance and sterile buffer solution. One preferred methodof sterilization may be by sterile filtration of a solution madeimmediately prior to use. The compound or related molecule may containpharmaceutically acceptable auxiliary substances as required toapproximate physiological conditions, such as buffering agents, tonicityadjusting agents and the like, for instance sodium acetate, sodiumlactate, sodium chloride, potassium chloride, calcium chloride, etc.

The stabilized Exendin-4 (1-39) compounds and related molecules of theinvention find use in a wide spectrum of applications. Some aredescribed in WO 99/40788 (relating to the inotropic and diuretic effectsof Exendin and GLP-1); and WO 98/39022 (relating to a method of sedatinga mammalian subject having increased activation of the central orperipheral nervous system comprising administering Exendin or GLP-1 oran agonist of Exendin or GLP-1 to the subject to produce a sedative oranxiolytic effect on the subject); and WO 93/18786 (relating to thetreatment of diabetes using GLP-1 (7-37) or GLP-1 (7-36)amide in aregimen which additionally comprises treatment with an oralhypoglycaemic agent, such as sulfonylurea, producing a strongsynergistic effect); and WO 98/19698 (relating to the use of GLP-1analogs for the regulation of obesity); WO 98/08531 (relating to the useof GLP-1 or analogs in a method of reducing mortality and morbidityafter myocardial infarction); WO 98/08873 (relating to the use of GLP-1or analogs in a method of attenuating post-surgical catabolic changesand hormonal responses to stress). Besides, the compounds of theinvention are suitable in a combination therapy with other antidiabeticagents, such as insulin, metformin, sulfonyl ureas andthiazolidinediones, or in combination therapy with other antiobesityagents, such as leptin, dexphenfluramine, amphetamine, etc.

Other formulations are within the scope of the present invention. Suchformulations include, but are not limited to, formulations that includeat least one of the stabilized Exendin-4 (1-39) compounds disclosedherein combined with liposomes, microspheres and liquid stabilizers.Depot formulations that include at least one of the stabilized Exendin-4(1-39) compounds are also envisioned. See U.S. Pat. Nos. 5,407,609 and5,654,008 for additional information.

A particular liquid formulation suitable for use with the presentstabilized Exendin-4 (1-39) compounds includes: about 50 mM histidine,about 100 to 200 mM sucrose, mannitol or other acceptable sugar, 20 mMmethionine, 20 mM Asparagine-glutamine or Asp, at a pH of about 5.3. Thecompound can be dissolved in nearly any suitable amount including about50 micrograms/mL to about 2.5 mg/mL

In one embodiment, the pharmaceutically acceptable compositionsdisclosed herein can include at least one of the following sequences:des Pro³⁶[Asp²⁸]Exendin-4 (1-39) (SEQ ID NO:15), desPro³⁶[IsoAsp²⁸]Exendin-4 (1-39) (SEQ ID NO:16), des Pro³⁶[Cyclicimide²⁸]Exendin-4 (1-39) (SEQ ID NO:17), des Pro³⁶[Met(O)¹⁴]Exendin-4(1-39) (SEQ ID NO:18), des Pro³⁶[Trp(O₂)²⁵]Exendin-4 (1-39) (SEQ IDNO:19), des Pro³⁶[Met(O)¹⁴, Asp²⁸]Exendin-4 (1-39) (SEQ ID NO:20), despro³⁶[Met(O)¹⁴, IsoAsp²⁸]Exendin-4 (1-39) (SEQ ID NO:21), desPro³⁶[Met(O)¹⁴, Cyclic imide²⁸]Exendin-4 (1-39) (SEQ ID NO:22), desPro³⁶[Met(O)¹⁴, Trp(O₂)²⁵]Exendin-4 (1-39) (SEQ ID NO:23), desPro³⁶[Trp(O₂)²⁵, Asp²⁸]Exendin-4 (1-39) (SEQ ID NO:98), desPro³⁶[Trp(O₂)²⁵, IsoAsp²⁸]Exendin-4 (1-39) (SEQ ID NO:99), desPro³⁶[Trp(O₂)²⁵, Cyclic imide²⁸]Exendin-4 (1-39) (SEQ ID NO:100), desPro³⁶[Met(O)¹⁴, Trp(O₂)²⁵]Exendin-4 (1-39) (SEQ ID NO:23), desPro³⁶[Met(O)¹⁴, Trp(O₂)²⁵, Asp²⁸]Exendin-4 (1-39) (SEQ ID NO:24), desPro³⁶[Met(O)¹⁴, Trp(O₂)²⁵, IsoAsp²⁸]Exendin-4 (1-39) (SEQ ID NO:25), anddes Pro³⁶[Met(O)¹⁴, Trp(O₂)²⁵, Cyclic imide²⁸]Exendin-4 (1-39) (SEQ IDNO:26), or

a pharmaceutically acceptable salt or solvate thereof.

Each of the specified compounds may optionally include the followinggroup linked to the N- or C-terminus thereof, preferably the C-terminus:-Lys₆-NH₂ (SEQ ID NO:106).

Additionally specific pharmaceutically acceptable compositions accordingto the invention include at least one of the following compounds:

[Asp²⁸]Exendin-4 (1-39) (SEQ ID NO:83),

[IsoAsp²⁸]Exendin-4 (1-39) (SEQ ID NO:84),

[Cyclic imide²⁸]Exendin-4 (1-39) (SEQ ID NO:85),

[Met(O)¹⁴]Exendin-4 (1-39) (SEQ ID NO:86),

[Trp(O₂)²⁵]Exendin-4 (1-39) (SEQ ID NO:87),

[Met(O)¹⁴, Asp²⁸]Exendin-4 (1-39) (SEQ ID NO:88)

[Met(O)¹⁴, IsoAsp²⁸]Exendin-4 (1-39) (SEQ ID NO:89),

[Met(O)¹⁴, Cyclic imide²⁸]Exendin-4 (1-39) (SEQ ID NO:90),

[Met(O)¹⁴, Trp(O₂)²⁵]Exendin-4 (1-39) (SEQ ID NO:91)

[Trp(O₂)²⁵, Asp²⁸]Exendin-4 (1-39) (SEQ ID NO:92),

[Trp(O₂)²⁵, IsoAsp²⁸]Exendin-4 (1-39) (SEQ ID NO:93),

[Trp(O₂)²⁵, Cyclic imide²⁸]Exendin-4 (1-39) (SEQ ID NO:94),

Met(O)¹⁴, Trp(O₂)²⁵, Asp²⁸]Exendin-4 (1-39) (SEQ ID NO:95),

[Met(O)¹⁴, Trp(O₂)²⁵, IsoAsp²⁸]Exendin-4 (1-39) (SEQ ID NO:96); and

[Met(O)¹⁴, Trp(O₂)²⁵, Cyclic imide²⁸]Exendin-4 (1-39) (SEQ ID NO:97)

or a pharmaceutically acceptable salt or solvate thereof.

In one embodiment, the pharmaceutically acceptable composition featuresa weight ratio of any one of the stabilized Exendin-4 (1-39) compoundsor related molecules described herein to Exendin-4 (1-39) or a variant,analogue or derivative thereof being less than about 50% (w/w), lessthan about 10% (w/w), or in some instances less than about 1% (w/w). Anappropriate weight ratio will depend on intended use of the compositionand other parameters such as the degree of stability needed.

The present stabilized Exendin-4 (1-39) compositions can be readily madeusing one or a combination of standard techniques. In one such approachthe methods include making/obtaining Exendin-4 (1-39) or a variant,analogue, or derivative thereof; and subsequently exposing or incubatingthe Exendin-4 (1-39) or the variant, analogue, or derivative thereofunder conditions sufficient to introduce at least one of the followingamino acids therein:

i) an Asn residue having a deaminated side chain, an Asn residue havinghydrolyzed side chain or a structural isomer of an Asp residue, whereinthe Asn or Asp residue corresponds to position 28 of Exendin-4,

ii) an oxidized methionine residue corresponding to position 14 ofExendin-4,

iii) an oxidized tryptophan residue corresponding to position 25 ofExendin-4,

iv) a deaminated or hydrolyzed Gln corresponding to position 13 ofExendin-4; and a pharmaceutically acceptable salt or solvate thereof.

Alternatively, the stabilized Exendin-4 (1-39) compounds of theinvention can be made by conventional peptide synthetic routes includinguse of the Merrifield synthesis. Modified amino acid residues in accordwith the invention can be purchased from commercial suppliers (eg.,modified Gln and Asp) or they can be readily made using standardtechniques (eg., oxidation of Met and Trp). See Merrifield, B. (1985) inScience 232:341.

Preferred practice of the methods further include the step of detectingpresence or absence of at least one of amino acids (i)-(iv). Preferablyalso such methods include the step of identifying at least one of theamino acids (i)-(iv) in the composition.

Suitable methods for detecting the stabilized Exendin-4 (1-39)compositions and related molecules disclosed herein are known in thefield and include, but are not limited to, reverse phase highperformance liquid chromatography (RP-HPLC), and liquidchromatography/mass spectrometry (LC-MS). Additionally suitabletechniques include conventional amino acid sequencing, peptide mapping,MS/MS and fluorescence.

In another aspect, the invention provides a method of making thepharmaceutically acceptable compositions that include stabilizedExendin-4 compositions or related molecules. In one embodiment, themethod includes at least one of the following steps:

a) obtaining Exendin-4 (1-39) or a variant, analogue or derivativethereof,

b) contacting the Exendin-4 (1-39) or the variant, analogue orderivative with at least one pharmaceutically acceptable carrier orvehicle to produce a mixture; and

c) incubating the mixture under conditions sufficient to introduce atleast one of the following amino acids therein:

i) an Asn residue having a deaminated side chain, an Asn residue havinghydrolyzed side chain or a structural isomer of an Asp residue, whereinthe Asn or Asp residue corresponds to position 28 of Exendin-4,

ii) an oxidized methionine residue corresponding to position 14 ofExendin-4,

iii) an oxidized tryptophan residue corresponding to position 25 ofExendin-4

iv) a deaminated or hydrolyzed Gln corresponding to position 13 ofExendin-4; and a pharmaceutically acceptable salt or solvate thereof.

Preferred practice of the methods further include the step of detectingpresence or absence of at least one of amino acids (i)-(iv). Preferablyalso such methods include the step of identifying at least one of theamino acids (i)-(iv) in the composition.

Typical conditions for making the stabilized Exendin-4 (1-39) compoundsand related molecules will be generally sufficient to introduce at leastone of the foregoing amino acid modifications (i)-(iii) as describedabove. Such conditions are preferably also capable of modifying theglutamine position 13 in the Exendin-4 molecule. Examples of suchconditions include, but are not limited to, exposure to water, buffer,heat, water vapor, oxygen, light, metals and metal ions. Such conditionscan include contact with a variety of temperatures including contactwith about 1° C. up to about 80° C., preferably 5° C. to about 45° C.Room temperature (25° C.) is generally preferred for some applications.Air has been found to be particularly good at modifying the amino acidresidues of the Exendin-4 molecule.

However as discussed, it will often be generally preferred to make thestabilized Exendin-4 (1-39) molecules by a synthetic or semi-syntheticapproach. A preferred example of such a strategy is use of theMerrifield peptide synthesis procedure in an automated format.

As discussed, the invention further provides a method of stabilizingExendin-4 (1-39) or a variant, analogue or derivative thereof fromdegradation before, during or after intended use. Typical methodsinclude at least one of the following steps:

a) obtaining Exendin-4 (1-39) or a variant, derivative or analoguethereof, and

b) incubating the Exendin-4 (1-39) or the variant, derivative oranalogue under conditions sufficient to introduce at least one of thefollowing amino acid residues therein:

-   -   i) an Asn residue having a deaminated side chain, an Asn residue        having hydrolyzed side chain or a structural isomer of an Asp        residue, wherein the Asn or Asp residue corresponds to position        28 of Exendin-4,    -   ii) an oxidized methionine residue corresponding to position 14        of Exendin-4,    -   iii) an oxidized tryptophan residue corresponding to position 25        of Exendin-4    -   iv) a deaminated or hydrolyzed Gln corresponding to position 13        of Exendin-4; and a pharmaceutically acceptable salt or solvate        thereof.

For instance, the method conditions include contact with at least one ofwater, heat, water vapor, light metal, metal ion or oxygen. Suchconditions can include contact with a variety of temperatures includingcontact with about 1° C. up to about 80° C., preferably 5° C. to about45° C. Room temperature (25° C.) is generally preferred for someapplications.

Also preferably, the method includes the step of identifying at leastone of the amino acids (i)-(iv) in the stabilized Exendin-4 (1-39) orvariant, analogue or derivative thereof. Optionally, the method mayfurther include contacting the stabilized Exendin-4 (1-39) or variant,derivative or analogue thereof with at least one pharmaceuticallyacceptable carrier or vehicle

As discussed, many suitable Exendin-4 and related compounds have beendisclosed PCT/DK00/00393, EP application 99610043.4 and U.S. provisionalapplication 60/143,591.

As disclosed in the PCT/DK00/00393 application, one type of Exendin-4compound is directed to a peptide conjugate comprising a peptide Xselected from the group consisting of

(a) an Exendin having at least 90% homology to Exendin-4;

(b) a variant of said Exendin wherein said variant comprises amodification selected from the group consisting of between one and fivedeletions at positions 34-39 and contains a Lys at position 40 having alipophilic substituent; or

(c) GLP-1 (7-36) or GLP-1 (7-37) having at least one modificationselected from the group consisting of:

-   -   (i) substitution of D-alanine, glycine or alpha-amino isobutyric        acid for alanine at position 8 and    -   (ii) a lipophilic substituent,

and Z, a peptide sequence of 4-20 amino acid units covalently bound tosaid variant, wherein each amino acid unit in said peptide sequence, Zis selected from the group consisting of Ala, Leu, Ser, Thr, Tyr, Asn,Gln, Asp, Glu, Lys, Arg, His, Met, Orn, and amino acid units of thegeneral formula I

—NH—C(R¹)(R²)—C(═O)—  (I)

wherein R¹ and R² are selected from the group consisting of hydrogen,C₁₋₆-alkyl, phenyl, and phenyl-methyl, wherein C₁₋₆-alkyl is optionallysubstituted with from one to three substituents selected from halogen,hydroxy, amino, cyano, nitro, sulfono, and carboxy, and phenyl andphenyl-methyl is optionally substituted with from one to threesubstituents selected from C₁₋₆-alkyl, C₂₋₆-alkenyl, halogen, hydroxy,amino, cyano, nitro, sulfono, and carboxy, or R¹ and R² together withthe carbon atom to which they are bound form a cyclopentyl, cyclohexyl,or cycloheptyl ring, e.g. 2,4-diaminobutanoic acid and2,3-diaminopropanoic acid, with the proviso that X is not Exendin-4 orExendin-3.

The peptide X is further characterised in being effective in improvingglucose tolerance in a diabetic mammal.

Furthermore, the PCT/DK00/00393 application features a novel variant ofa parent Exendin, wherein said parent Exendin has an amino acid sequencehaving at least an 90% homology to Exendin-4 and wherein said variantlowers the blood glucose level in a mammal, binds to a GLP-1 receptorand has at least one modification selected from the group consisting of(a) between one and five deletions at positions 34-38, and (b) containsa Lys at position 40 having a lipophilic substituent attached to theepsilon amino group of said lysine.

By the phrase “Exendin variant” is meant a variant of a parent Exendinpeptide having at least about 90% homology, more preferably at leastabout 95% homology to Exendin-4, which have Exendin activity, e.g.,lowers the blood glucose level in a mammal and binds to a GLP-1receptor. In a preferred embodiment the parent Exendin peptide has anamino acid sequence which differs by five amino acids, preferably byfour amino acids, more preferably by three amino acids, even morepreferably by two amino acids, and still more preferably by one aminoacid residue from the amino acid sequence of Exendin-4(1-39). See thePCT /DK00/00393 application for additional information.

Additionally suitable Exendin-4 variants, analogues and derivativesthereof have been disclosed in the following references, each of whichis individually incorporated by reference: U.S. Pat. Nos. 6,358,924;6,344,180; 6,284,725; 6,277,819; 6,271,241; 6,268,343; 6,191,102;6,051,689; 6,006,753; 5,846,937; 5,670,360; 5,614,492; 5,846,937;5,545,618; 6,410,508; 6,388,053; 6,384,016; 6,329,336; 6,110,703,5,846,747; 5,670,360; 5,631,224; 5,424,286; WO 98/05351; WO 98/30231; WO99/07404, WO 99/25727; WO 99/25728; or WO 99/46283. Each of saidcompounds can be stabilized in accord with this invention eg., byexposing the compounds to the stabilizing conditions disclosed herein.Alternatively, such stabilized Exendin-4 (1-39) compounds can be madesynthetically using the Merrifield synthesis and starting materials thatcan be purchased or readily made.

It should be understood that the compositions and compounds of theinvention might also be in the preferred amide (NH₂) or in the free acid(OH) form or in the form of a salt thereof.

In embodiments in which one or more of the stabilized Exendin-4 (1-39)compounds and related molecules are used therapuetically, such use willtypically involve administration of one or more of the pharmaceuticallyacceptable compositions disclosed herein. Such a composition can becombined with at least one of Exendin-4, Exendin-3, or derivatives,analogs or variants thereof together with a suitable amount of vehicleand/or stabilizer. In one embodiment, such an approach involvesadministering the composition (eg. as a depot formulation, liquidformulation, with microspheres or liposomes, i.v) to provide a dosage ofabout 0.1 pg/kg, to 1.000 mg/kg body weight. The amount of thecomposition to use will depend on recognized parameters including age,severity of the disease, total body weight, sex and other factors.

A “peptide” as used herein is any compound produced by amide formationbetween a carboxyl group of one amino acid and an amino group ofanother. The amide bonds in peptides may be called peptide bonds. Theword peptide usually applies to compounds whose amide bonds are formedbetween C-1 of one amino acid and N-2 of another (sometimes calledeupeptide bonds), but it includes compounds with residues linked byother amide bonds (sometimes called isopeptide bonds). Peptides withfewer than about 10-20 residues may also be called oligopeptides andpeptides with more than 20 residues are called polypeptides.Polypeptides of specific sequences of more than about 50 residues areusually known as proteins. A “natural polypeptide sequence” as usedherein refers to a polypeptide sequence consisting of natural L-aminoacid residues and which is capable of being expressed by a recombinanthost cell. The X compounds herein are all peptide sequences of 40 aminoacid residues or less.

“GLP-1” as used herein includes GLP-1 (7-37)-OH, GLP-1 (7-37)-NH₂, GLP-1(7-36)-OH, and GLP-1 (7-36)-NH₂.

Molecules are “related” to Exendin-4 if they are recognized analogues,derivatives, or variants thereof as described herein and in thePCT/DK00/00393, EP application 99610043.4, and U.S. provisionalapplication 60/143,591. Other such molecules have been disclosed in thefollowing U.S. Pat. Nos. 6,358,924; 6,344,180; 6,284,725; 6,277,819;6,271,241; 6,268,343; 6,191,102; 6,051,689; 6,006,753; 5,846,937;5,670,360; 5,614,492; 5,846,937; 5,545,618; 6,410,508; 6,388,053;6,384,016; 6,329,336; 6,110,703, 5,846,747; 5,670,360; 5,631,224.5,424,286; WO 98/05351; WO 98/30231; WO 99/07404, WO 99/25727; WO99/25728; and WO 99/46283, for example.

“Agonist” refers to an endogenous substance or a drug that can interactwith a receptor and initiate a physiological or a pharmacologicalresponse characteristic of that receptor (contraction, relaxation,secretion, enzyme activation, etc.).

“Antagonist” refers to a drug or a compound that opposes thephysiological effects of another. At the receptor level, it is achemical entity that opposes the receptor-associated responses normallyinduced by another bioactive agent.

“Partial agonist” refers to an agonist, which is unable to inducemaximal activation of a receptor population, regardless of the amount ofdrug applied. A “partial agonist” may be termed “agonist withintermediate intrinsic efficacy” in a given tissue. Moreover, a partialagonist may antagonize the effect of a full agonist that acts on thesame receptor.

“Receptor” refers to a molecule or a polymeric structure in or on a cellthat specifically recognizes and binds a compound acting as a molecularmessenger (neurotransmitter, hormone, lymphokine, lectin, drug, etc.).

By “Exendin variant” of the present invention is to be understood avariant of a parent Exendin peptide having at least about 90% homologyto Exendin-4 and most preferably having at least about 95% homology toExendin-4(1-39), which has Exendin activity, e.g., lowers the bloodglucose level in a mammal and binds to a GLP-1 receptor.

“Exendin-4” as used herein refers to Exendin-4(1-39) the amino acidsequence of which is disclosed in U.S. Pat. No. 5,424,286, SEQ ID NO:2,and Exendin-4(1-40) as disclosed by Chen & Drucker in The Journal ofBiological Chemistry, Vol. 272, No. 7, pp. 4108-15 which differs only inhaving glycine in position 40 as C-terminal amino acid residue. Thehomology of the parent Exendin is determined as the degree of identitybetween two protein sequences indicating a derivation of the firstsequence from the second. The homology may suitably be determined bymeans of computer programs known in the art such as GAP provided in theGCG program package (Program Manual for the Wisconsin Package, Version8, August 1994, Genetics Computer Group, 575 Science Drive, Madison,Wis., USA 53711) (Needleman, S. B. and Wunsch, C.D., (1970), J. Mol.Biol. 48:443-453). The following settings for polypeptide sequencecomparison may be used: GAP creation penalty of 3.0 and GAP extensionpenalty of 0.1. By “Met(O)” of the present invention is intended to meana methionine sulfoxide or methionine sulfone.

By “Trp(O₂)” of the present invention is intended to meanN-formylkynurenine, a tryptophan residue that has undergone dioxidation.

“Salts” include pharmaceutically acceptable salts, such as acid additionsalts and basic salts. Examples of acid addition salts are hydrochloridesalts, sodium salts, hydrobromide salts, etc. Examples of basic saltsare salts where the cation is selected from alkali metals, such assodium and potassium, alkaline earth metals, such as calcium, andammonium ions ⁺N(R³)₃(R⁴), where R³ and R⁴ independently designatesoptionally substituted C₁₋₆-alkyl, optionally substituted C₂₋₆-alkenyl,optionally substituted aryl, or optionally substituted heteroaryl. Otherexamples of pharmaceutically acceptable salts are; e.g., those describedin “Remington's Pharmaceutical Sciences” 17. Ed. Alfonso R. Gennaro(Ed.), Mark Publishing Company, Easton, Pa., U.S.A., 1985 and morerecent editions, and in Encyclopedia of Pharmaceutical Technology.

Single and triple letter designations for the amino acids are usedinterchangeably. For example, it will be appreciated that Lys and Krefer to lysine, Asp and D refer to aspartic acid, Glu and E refer toglutamic acid, etc. A complete description of the amino aciddesignations can be found in Alberts, B. et al. in Molecular Biology ofthe Cell, 2^(nd) Ed. Garland Publishing, Inc. (New York) (1989).

The following Examples are illustrative and not limiting as to the scopeof the present invention.

Example 1 Stabilization of Exendin-4 and Related Molecules by StructuralIsomerization and/or Oxidation

Pharmaceutical compositions of Exendin-4(1-39) or a variant, analogue,or derivative thereof or aqueous solutions of Exendin-4(1-39) or avariant, analogue, or derivative thereof can be stabilized by oxidationor functional isomerization at various points in the Exendin-4(1-39)sequence.

Storage of the Exendin-4(1-39) or a variant, analogue, or derivativethereof in an aqueous solution at a temperature of between about 0° C.and about 50° C., more particularly between about 4° C. and roomtemperature can induce a structural rearrangement at the28-L-Asparaginyl residue of the Exendin-4(1-39) peptide. Suitableaqueous solutions are not particularly limited and may include aqueouspharmaceutical compositions which may have one or more additionaladditives to facilitate administration to a patient or to stabilize orsolublize the Exendin-4(1-39) or a variant, analogue, or derivativethereof.

Although not bound by theory, one synthetic pathway through which thestructural rearrangement of the 28-L-Asparaginyl residue (structure A)may occur is presented in Synthetic Scheme 1. The 28-L-Asparaginylresidue undergoes deamination in aqueous media to form a Cyclic imideresidue, which is depicted as (structure B). Reversible hydrolysis ofthe Cyclic imide leads to formation of both L-aspartyl residue(structure C) or a L-isoAspartyl residue (structure D). Each hydrolysisreaction is reversible such that one or more stabilized Exendincompounds having the 28-Cyclic imide residue, 28-L-aspartyl residue or28-L-isoAspartyl residue may be present in the aqueous solution.

Oxidation products are formed by incubating an aqueous solution ofExendin-4 (1-39) or an Exendin-4 (1-39) compound in the presence of anoxidant such as dioxygen.

Typically pharmaceutical compositions of the present invention, whichare exposed to molecular dioxygen during processing, or storage aresusceptible to oxidation of the 14-methionine or 25-tryptophan residues.

One non-limiting route to oxidation of the 14-methionine residue occursupon oxidation in the presence of molecular dioxygen. A 14-methioninesulfoxide is produced by reaction of Exendin-4(1-39) in aqueous solutionwith half an equivalent of molecular dioxygen. Further exposure of thestabilized Exendin compound having a 14-methionine sulfoxide results information of 14-methionine sulfone in which the 14-methionine residuehas been oxidized by one equivalent of dioxygen.

The 25-tryptophan residue of Exendin-4(1-39) or an Exendin-4(1-39)variant, analogue, or derivative may be oxidized by 0.5, 1, 1.5, 2 ormore equivalents of molecular dioxygen. Common oxidized tryptophanproducts include hydroxytryptophan, N-formylkynuranine, kynuranine, and3-hydroxykynuranine. Under normal storage or incubation conditions ofabout 0° C. to about 25° C., one equivalent of molecular dioxygenoxidizes the 25-tryptophan residue of Exendin-4(1-39) or anExendin-4(1-39) variant to form an Exendin-4(1-39) compound having a25-N-formylkynuranine residue.

Example 2 Synthesis of Compound 5, 6 and 14 (Stabilized Derivatives ofCompound 1)

Compound 1 (des Pro³⁶ Exendin-4 (1-39)-K₆; SEQ ID NO:1) has thestructure shown in FIG. 1 and it was made using the Merrifieldtechnique. See the PCT/DK00/00393 application, for example, for moreinformation.

About 458 mg of Compound 1 was dissolved in 100 mM NH₄HCO₃ pH 7.9 to aconcentration of 10 mg/mL. The solution was incubated at 40° C. for 6days to yield approx. 20% Compound 5, 10% Compound 14 and 50% Compound6.

The stabilized product Compound 5 and Compound 6 can be purified bypreparative RP-HPLC isocratic elution or using a gradient, respectively.Identification is accomplished by relative retention time in combinationwith Amino acid Sequencing and LC-MS (ESI⁺/TOF)

Example 3 Synthesis of a Compound 7 (Stabilized Derivative of Compound1)

About 424 mg of Compound 1 was dissolved in 100 mM NH₄HCO₃ pH 7.9 to aconcentration of 10 mg/mL. The solution was incubated at 40° C. for 5days to yield approx. 20% Compound 5, 60% Compound 6. Compound 6 waspurified by preparative RP-HPLC. Approximately 100 mg Compound 6 wasobtained and lyophilised. About 100 mg Compound 6 was reconstituted in100 mM NaH₂PO₄ buffer and adjusted to pH 5.3 with NaOH to aconcentration of 5 mg/mL. The solution was incubated at 40° C. for 5days to yield approximately 40% Compound 7.

The stabilized product Compound 7 can be purified by preparative RP-HPLCgradient elution. Identification is accomplished by relative retentiontime in combination with LC-MS (ESI⁺/TOF).

Example 4 Synthesis of Compounds 3 and 4 (A Stabilized Derivative ofCompound 1)

Compound 1 was made as described. It was dissolved (10 mg) in 50 mMcitrate buffer containing 3% (w/v) D-mannitol to a concentration of 100μg/mL. The solution was incubated at 25° C. for at least 6 days andexposed to light 350 nm mean wavelength.

The stabilized products Compound 3 and 4 can be purified by preparativeRP-HPLC. Identification is accomplished by relative retention time incombination with peptide mapping by LC-MS (ESI+/TOF) of a tryptic digestand MS/MS plus flourescence detection.

Example 5 Synthesis of Compound 2 (Stabilized Derivative of Compound 1)

Compound 1 was made as described. It was dissolved (100 mg) in 10 mLpure water to a concentration of 10 mg/mL and exposed to H₂O₂ by adding2.3 mL of 3.5% H₂O₂. The solution was incubated at 25° C. for 1 day toyield approx. 100% Compound 2.

The stabilized product Compound 2 can be purified by preparativeRP-HPLC. Identification is accomplished by relative retention time incombination with peptide mapping by LC-MS (ESI⁺/TOF) of a tryptic digestand MS/MS plus flourescence detection.

Example 6 Peptide Synthesis

Peptide synthesis of Compound 8,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Glu-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH₂([Glu¹³]Exendin-4-NH₂; SEQ ID NO:8) on TentaGel S-RAM)

Dry TentaGel S-RAM resin (0.23 mmol/g, 1.0 g) is placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand swelled for two hours in DMF (5 ml). The Fmoc group is removedaccording to the procedure described above, and the peptide according tothe sequence is assembled as described under “Batchwise peptidesynthesis on TentaGel S-RAM resins [Due Larsen, B. and Holm, A. (1998)J. Pept. Res. 52, 470]. After completion of the synthesis, thepeptide-resin is washed with DMF (3×5 ml, 1 min each), DCM (3×5 ml, 1min each), diethyl ether (3×5 ml, 1 min each) and dried in vacuo. Thepeptide is cleaved from the resin by treatment with 95% trifluoroaceticacid and 5% ethanedithiol v/v at r.t. for 2 h. The filtered resins arewashed with 95% TFA-water and filtrates and washings are diluted byadding water. The resulting mixture is extracted 3 times with ether andfinally freeze dried. The crude peptide is purified by preparative HPLC.The purified product was found to be homogeneous and the purity wasfound to be better than 95%. The identity of the peptide was confirmedby ES-MS (Mw found 4185.17 and calculated 4185.01).

Peptide synthesis of Compound 9,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Glu-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH₂([Glu¹³, Asp²⁸]Exendin-4-NH₂; SEQ ID NO:9) on TentaGel S-RAM.

Dry TentaGel S-RAM resin (0.23 mmol/g, 1.0 g) is placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand swelled for two hours in DMF (5 ml). The Fmoc group is removedaccording to the procedure described above, and the peptide according tothe sequence is assembled as described under “Batchwise peptidesynthesis on TentaGel S-RAM resins [Due Larsen, B. and Holm, A. (1998)J. Pept. Res. 52, 470]. After completion of the synthesis, thepeptide-resin is washed with DMF (3×5 ml, 1 min each), DCM (3×5 ml, 1min each), diethyl ether (3×5 ml, 1 min each) and dried in vacuo. Thepeptide is cleaved from the resin by treatment with 95% trifluoroaceticacid and 5% ethanedithiol v/v at r.t. for 2 h. The filtered resins arewashed with 95% TFA-water and filtrates and washings are diluted byadding water. The resulting mixture is extracted 3 times with ether andfinally freeze dried. The crude peptide is purified by preparative HPLC.The purified product was found to be homogeneous and the purity wasfound to be better than 95%. The identity of the peptide was confirmedby ES-MS (Mw found 4186.25 and calculated 4186.18).

Peptide synthesis of Compound 10,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asp-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH₂([Asp²⁸]Exendin-4-NH₂; SEQ ID NO:10) on TentaGel S-RAM.

Dry TentaGel S-RAM resin (0.23 mmol/g, 1.0 g) is placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand swelled for two hours in DMF (5 ml). The Fmoc group is removedaccording to the procedure described above, and the peptide according tothe sequence is assembled as described under “Batchwise peptidesynthesis on TentaGel S-RAM resins [Due Larsen, B. and Holm, A. (1998)J. Pept. Res. 52, 470]. After completion of the synthesis, thepeptide-resin is washed with DMF (3×5 ml, 1 min each), DCM (3×5 ml, 1min each), diethyl ether (3×5 ml, 1 min each) and dried in vacuo. Thepeptide is cleaved from the resin by treatment with 95% trifluoroaceticacid and 5% ethanedithiol v/v at r.t. for 2 h. The filtered resins arewashed with 95% TFA-water and filtrates and washings are diluted byadding water. The resulting mixture is extracted 3 times with ether andfinally freeze dried. The crude peptide is purified by preparative HPLC.The purified product was found to be homogeneous and the purity wasfound to be better than 95%. The identity of the peptide was confirmedby ES-MS (Mw found 4185.38 and calculated 4185.01).

Peptide synthesis of Compound 11,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Glu-Met-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-NH₂(des Pro³⁶-[Glu¹³]Exendin-4-(Lys)₆-NH₂; SEQ ID NO:11) on TentaGel S-RAM.

Dry TentaGel S-RAM resin (0.23 mmol/g, 1.0 g) is placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand swelled for two hours in DMF (5 ml). The Fmoc group is removedaccording to the procedure described above, and the peptide according tothe sequence is assembled as described under “Batchwise peptidesynthesis on TentaGel S-RAM resins [Due Larsen, B. and Holm, A. (1998)J. Pept. Res. 52, 470]. After completion of the synthesis, thepeptide-resin is washed with DMF (3×5 ml, 1 min each), DCM (3×5 ml, 1min each), diethyl ether (3×5 ml, 1 min each) and dried in vacuo. Thepeptide is cleaved from the resin by treatment with 95% trifluoroaceticacid and 5% ethanedithiol v/v at r.t. for 2 h. The filtered resins arewashed with 95% TFA-water and filtrates and washings are diluted byadding water. The resulting mixture is extracted 3 times with ether andfinally freeze dried. The crude peptide is purified by preparative HPLC.The purified product was found to be homogeneous and the purity wasfound to be better than 95%. The identity of the peptide was confirmedby ES-MS (Mw found 4855.72 and calculated 4855.52).

Peptide synthesis of Compound 12,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met(O)-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-NH₂(des Pro³⁶-[Met(O)¹⁴]Exendin-4-(Lys)₆-NH₂; SEQ ID NO:12) on TentaGelS-RAM.

Dry TentaGel S-RAM resin (0.23 mmol/g, 1.0 g) is placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand swelled for two hours in DMF (5 ml). The Fmoc group is removedaccording to the procedure described above, and the peptide according tothe sequence is assembled as described under “Batchwise peptidesynthesis on TentaGel S-RAM resins [Due Larsen, B. and Holm, A. (1998)J. Pept. Res. 52, 470]. The Methionine sulfoxide was incorporated asFmoc-Met(O)—OH (Purchased from Bachem) according to the describedcoupling procedures [Due Larsen, B. and Holm, A. (1998) J. Pept. Res.52, 470]. After completion of the synthesis, the peptide-resin is washedwith DMF (3×5 ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether(3×5 ml, 1 min each) and dried in vacuo. The peptide is cleaved from theresin by treatment with 95% trifluoroacetic acid and 5% ethanedithiolv/v at r.t. for 2 h. The filtered resins are washed with 95% TFA-waterand filtrates and washings are diluted by adding water. The resultingmixture is extracted 3 times with ether and finally freeze dried. Thecrude peptide is purified by preparative HPLC. The purified product wasfound to be homogeneous and the purity was found to be better than 95%.The identity of the peptide was confirmed by ES-MS (Mw found 4870.88 andcalculated 4870.53).

Peptide synthesis of Compound 13,H-His-Gly-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Leu-Ser-Lys-Gln-Met(O₂)-Glu-Glu-Glu-Ala-Val-Arg-Leu-Phe-Ile-Glu-Trp-Leu-Lys-Asn-Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Ser-Lys-Lys-Lys-Lys-Lys-Lys-NH₂(des Pro³⁶-[Met(O₂)¹⁴]Exendin-4-(Lys)₆-NH₂; SEQ ID NO:13) on TentaGelS-RAM.

Dry TentaGel S-RAM resin (0.23 mmol/g, 1.0 g) is placed in apolyethylene vessel equipped with a polypropylene filter for filtrationand swelled for two hours in DMF (5 ml). The Fmoc group is removedaccording to the procedure described above, and the peptide according tothe sequence is assembled as described under “Batchwise peptidesynthesis on TentaGel S-RAM resins [Due Larsen, B. and Holm, A. (1998)J. Pept. Res. 52, 470]. The Methionine sulfone was incorporated asFmoc-Met(O₂)—OH (Purchased from Bachem) according to the describedcoupling procedures [Due Larsen, B. and Holm, A. (1998) J. Pept. Res.52, 470]. After completion of the synthesis, the peptide-resin is washedwith DMF (3×5 ml, 1 min each), DCM (3×5 ml, 1 min each), diethyl ether(3×5 ml, 1 min each) and dried in vacuo. The peptide is cleaved from theresin by treatment with 95% trifluoroacetic acid and 5% ethanedithiolv/v at r.t. for 2 h. The filtered resins are washed with 95% TFA-waterand filtrates and washings are diluted by adding water. The resultingmixture is extracted 3 times with ether and finally freeze dried. Thecrude peptide is purified by preparative HPLC. The purified product wasfound to be homogeneous and the purity was found to be better than 95%.The identity of the peptide was confirmed by ES-MS (Mw found 4886.68 andcalculated 4886.53).

Oral Glucose Tolerance Test (OGTT)

The antidiabetic effect of the present compounds was tested in an oralglucose tolerance test (OGTT). In short, animals were fasted over night,and blood samples were taken from the tip of the tail, and the bloodglucose measured. The whole blood glucose (mM) concentration wasanalysed by the immobilised glucose oxidase method using a drop of blood(<5 μl; Elite Autoanalyser, Bayer, Denmark) following the manufacturer'smanual. The animals were kept fasted throughout the test. Immediatelyafter the initial blood sample (fasting blood glucose level) testcompound or vehicle was administered i.p. Fifteen minutes later an oraldose of glucose ((1 g/kg) Sigma, St. Louis, Mo., U.S.A.)), dissolved inphosphate buffer (pH=7.40) was given, and the animals were returned totheir home cages (time=0). Blood glucose (BG) levels were measured attime=30 min, time=60 min and time=120 min for Compound 1, 8, 9, 10 and11 and additionally time=240 min for Compounds 1, 2, 5, 6, 7 and 14. Toanalyse the effects of the test compounds on oral glucose tolerance theabsolute difference in BG from baseline (fasting BG) was calculated foreach time point. The area under the curve (AUC) (FIG. 1-8) for the wholeexperiment (AUC₀₋₁₂₀ min) or (AUC₀₋₂₄₀ min) was determined using thetrapezoid method. Before starting the experimental series an OGTT wasused to stratify animals into groups of 9-10 animals displaying similarglucose tolerances in all groups. Animals displaying excessive glucoseexcursion 30 min after glucose loading (BG>33 mmol/l) were excluded fromthe study.

Results of the Glucose Tolerance Test

The results of the glucose tolerance test are illustrated in the FIGS.1-4. The calculated ED₅₀ values are presented in Table 1.

TABLE 1 shows the ED₅₀ based on curve fits and the standard deviationratio (SD_(r)) Major Compound in Test ED₅₀ Solution (nmol/kg) SD_(r)Compound 1 0.049 3.8 Compound 14 0.55 1.3 Compound 6 0.013 19 Compound 70.21 2.1

The fit of the data obtained after treatment with Compound 1 isillustrated in FIG. 1. The calculated ED₅₀ and SD_(r) is shown in Table1.

The fit of the data obtained after treatment with Compound 14 isillustrated in FIG. 2. The calculated ED₅₀ and SD_(r) is shown in Table1.

The fit of the data obtained after treatment with Compound 6 isillustrated in FIG. 3. The calculated ED₅₀ and SD_(r) is shown in Table1.

The fit of the data obtained after treatment with Compound 7 isillustrated in FIG. 4. The calculated ED₅₀ and SD_(r) is shown in Table1.

In another set of experiments Compound 1, Compound 2, and Compound 5were tested as described above. The results are illustrated in FIGS. 5,6 and 7. The ED₅₀ values are outlined in Table 2.

TABLE 2 ED₅₀ based on curve fits. The calculation of standard deviationratio (SD_(r)) is presented above. Major Compound in Test ED₅₀ Solution(nmol/kg) SD_(r) Compound 1 0.073 4.1 Compound 5 0.043 4.1 Compound 20.011 36

The fit of the data obtained after treatment with Compound 1 isillustrated in FIG. 5. The calculated ED₅₀ and SD_(r) is shown in Table2.

The fit of the data obtained after treatment with Compound 5 isillustrated in FIG. 6. The calculated ED₅₀ and SD_(r) is shown in Table2.

The fit of the data obtained after treatment with Compound 2 isillustrated in FIG. 7. The calculated ED₅₀, and SD_(r) are shown inTable 1.

In conclusion, the above Compounds all showed anti diabetic effects bysignificantly increased glucose tolerance in the db/db mice, as shown bya reduction in AUC_(0-120 min) or AUC_(0-240 min) (illustrated in FIGS.1-8). All the solutions tested had positive effect on glucose tolerancein doses above 0.01 nmol/kg. Thus, administration of the test compoundsproduced a dose-dependent lowering of blood glucose following a glucoseload.

The invention described and claimed herein is not to be limited in scopeby the specific embodiments herein disclosed, since these embodimentsare intended as illustrations of several aspects of the invention. Anyequivalent embodiments are intended to be within the scope of thisinvention. Indeed, various modifications of the invention in addition tothose shown and described herein will become apparent to those skilledin the art from the foregoing description. Such modifications are alsointended to fall within the scope of the appended claims. Variousreferences are cited herein, the disclosure of which are incorporated byreference in their entireties.

1. A composition comprising an exendin-4 (1-39) analog which comprisesat least one modification which is selected from the group consistingof: (i) an alpha-asparate (Asp) or beta-aspartate (isoaspartyl) orcyclic imide residue at a position corresponding to the Asn residue atposition 28 of exendin-4; (ii) an oxidized methionine residue at aposition corresponding to position 14 of exendin-4; (iii) an oxidizedtryptophan residue at a position corresponding to position 25 ofexendin-4; and (iv) a deamidated or hydrolyzed Gln at a positioncorresponding to position 13 of exendin-4; or a pharmaceuticallyacceptable salt or solvate thereof.
 2. The composition of claim 1,wherein the exendin-4 analog further comprises at least one peptidesequence Z of 4-20 amino acid residues covalently bound to thestabilized exendin.
 3. The composition of claim 1, wherein the exendin-4analog further comprises a deletion of 0 to 5 amino acids at positionscorresponding to position 34-38 of exendin-4.
 4. The composition ofclaim 1, wherein the oxidized methionine residue is selected from thegroup consisting of a methioninyl sulfoxide or a methioninyl sulfone. 5.The composition of claim 1, wherein the oxidized tryptophan residuecomprises an oxidized 3H-indol-3-yl group.
 6. The composition of claim1, wherein the oxidized tryptophan residue is selected from the groupconsisting of N-formylkynurenine (NFK), 3-hydroxykynurenine (3-OH-KYN),hydroxytryptophan (HTRP), and kynurenine (KYN).
 7. The composition ofclaim 1, wherein the cyclic imide residue is selected from the groupconsisting of an aspartimide and a glutimide.
 8. The composition ofclaim 1, wherein Z comprises between about 4 to about 20 Lys amino acidunits.
 9. The composition of claim 1, wherein Z comprises 6 Lys aminoacid units.
 10. The composition of claim 1, wherein Z is covalentlybound to the stabilized exendin-4 (1-39) analog at the C-terminalcarbonyl function.
 11. The composition of claim 10 further comprisingthe following group linked to the C-terminus of the compound: -Lys₆-NH₂(SEQ ID NO:106).
 12. The composition of claim 1, wherein the stabilizedexendin-4 (1-39) analog is selected from the group consisting of: desPro³⁶[Asp²⁸]Exendin-4 (1-39) (SEQ ID NO:15), desPro³⁶[IsoAsp²⁸]Exendin-4 (1-39) (SEQ ID NO:16), desPro³⁶[Met(O)¹⁴,Asp²⁸]Exendin-4 (1-39) (SEQ ID NO:20), desPro³⁶[Met(O)¹⁴,IsoAsp²⁸]Exendin-4 (1-39) (SEQ ID NO:21), desPro³⁶[Trp(O₂)²⁵,Asp²⁸]Exendin-4 (1-39) (SEQ ID NO:98), desPro³⁶[Trp(O₂)²⁵,IsoAsp²⁸]Exendin-4 (1-39) (SEQ ID NO:99), desPro³⁶[Met(O)¹⁴ Trp(O₂)²⁵ Asp²⁸]Exendin-4 (1-39) (SEQ ID NO:24), desPro³⁶[Met(O)¹⁴,Trp(O₂)²⁵,IsoAsp²⁸]Exendin-4 (1-39) (SEQ ID NO:25), desPro³⁶[Cyclic imide²⁸]Exendin-4 (1-39) (SEQ ID NO:17), desPro³⁶[Met(O)¹⁴,Cyclic imide²⁸]Exendin-4 (1-39) (SEQ ID NO:22), desPro³⁶[Met(O)¹⁴,Trp(O₂)²⁵,Cyclic imide²⁸]Exendin-4 (1-39) (SEQ ID NO:26),[Asp²⁸]Exendin-4 (1-39) (SEQ ID NO:83), [IsoAsp²⁸]Exendin-4 (1-39) (SEQID NO:84), [Cyclic imide²⁸]Exendin-4 (1-39) (SEQ ID NO:85),[Glu¹³,Asp²⁸]Exendin-4-NH₂ (SEQ ID NO:9), [Met(O)¹⁴,Asp²⁸]Exendin-4(1-39) (SEQ ID NO:88), [Met(O)¹⁴,IsoAsp²⁸]Exendin-4 (1-39) (SEQ IDNO:89), [Met(O)¹⁴,Cyclic imide²⁸]Exendin-4 (1-39) (SEQ ID NO:90),[Trp(O₂)²⁵,Asp²⁸]Exendin-4 (1-39) (SEQ ID NO:92),[Trp(O₂)²⁵,IsoAsp²⁸]Exendin-4 (1-39) (SEQ ID NO:93), [Trp(O₂)²⁵,Cyclicimide²⁸]Exendin-4 (1-39) (SEQ ID NO:94), [Met(O)¹⁴ Trp(O₂)²⁵Asp²⁸]Exendin-4 (1-39) (SEQ ID NO:95),[Met(O)¹⁴,Trp(O₂)²⁵,IsoAsp²⁸]Exendin-4 (1-39) (SEQ ID NO:96), and[Met(O)¹⁴,Trp(O₂)²⁵,Cyclic imide²⁸]Exendin-4 (1-39) (SEQ ID NO:97). or apharmaceutically acceptable salt or solvate thereof.
 13. The compositionof claim 1, wherein the stabilized exendin-4 (1-39) analog is selectedfrom the group consisting of: H-(Lys)₆-desPro³⁶[Asp²⁸]Exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO:27), H-(Lys)₆-desPro³⁶,Pro³⁷,Pro³⁸[Asp²⁸]Exendin-4(1-39)-NH₂ (SEQ ID NO:29),H-Asn-(Glu)₅-des Pro³⁶,Pro³⁷,Pro³⁸[Asp²⁸]Exendin-4(1-39)-NH₂ (SEQ IDNO:30), des Pro³⁶,Pro³⁷,Pro³⁸[Asp²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ IDNO:31), H-(Lys)₆-des Pro³⁶,Pro³⁷,Pro³⁸[Asp²⁸]Exendin-4(1-39)-(Lys)₆-NH₂(SEQ ID NO:32), H-Asn-(Glu)₅-desPro³⁶,Pro³⁷,Pro³¹[Asp²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:33),H-(Lys)₆-des Pro³⁶[Trp(O₂)²⁵,Asp²⁸]Exendin-4(1-39)-Lys₆-NH₂ (SEQ IDNO:41), H-(Lys)₆-desPro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵,Asp²⁸]Exendin-4(1-39)-NH₂ (SEQ ID NO:43),H-Asn-(Glu)₅-des Pro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵,Asp²⁸]Exendin-4(1-39)-NH₂(SEQ ID NO:44), desPro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵,Asp²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ IDNO:45), H-(Lys)₆-desPro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵,Asp²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ IDNO:46), H-Asn-(Glu)₅-desPro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵,Asp²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ IDNO:47), H-(Lys)₆-des Pro³⁶[Met(O)¹⁴,Asp²⁸]Exendin-4(1-39)-Lys₆-NH₂ (SEQID NO:55), H-(Lys)₆-desPro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]Exendin-4(1-39)-NH₂ (SEQ ID NO:57),H-Asn-(Glu)₅-des Pro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]Exendin-4(1-39)-NH₂(SEQ ID NO:58), desPro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ IDNO:59), H-(Lys)₆-des Pro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:60), H-Asn-(Glu)₅-desPro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Asp²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ IDNO:61), H-Lys₆-desPro³⁶[Met(O)¹⁴,Trp(O₂)²⁵,Asp²⁸]Exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO:69),H-(Lys)₆-desPro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Trp(O₂)²⁵,Asp²⁸]Exendin-4(1-39)-NH₂ (SEQ IDNO:71), H-Asn-(Glu)₅-desPro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Trp(O₂)²⁵,Asp²⁸]Exendin-4(1-39)-NH₂ (SEQ IDNO:72), desPro³⁶,Pro³⁷,Pro³⁸[Met(O)¹⁴,Trp(O₂)²⁵,Asp²⁸]Exendin-4(1-39)-(Lys)₆-NH₂(SEQ ID NO:73), H-(Lys)₆-des Pro³⁶,Pro³⁷,Pro³⁸[Met(O)1⁴,Trp(O₂)²⁵,Asp²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:74),H-Asn-(Glu)₅-des Pro³⁶, Pro³⁷, Pro³⁸[Met(O)¹⁴, Trp(O₂)²⁵,Asp²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:75), H-(Lys)₆-desPro³⁶[Cyclic imide²⁸]Exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO:34), desPro³⁶,Pro³⁷,Pro³⁸[Cyclic imide²⁸]Exendin-4(1-39)-NH₂ (SEQ ID NO:35),H-(Lys)₆-des Pro³⁶,Pro³⁷,Pro³⁸[Cyclic imide²⁸]Exendin-4(1-39)-NH₂ (SEQID NO:36), H-Asn-(Glu)₅-des Pro³⁶,Pro³⁷,Pro³⁸[Cyclicimide²⁸]Exendin-4(1-39)-NH₂ (SEQ ID NO:37), des Pro³⁶,Pro³⁷,Pro³⁸[Cyclicimide²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:38), H-(Lys)₆-desPro³⁶,Pro³⁷,Pro³⁸[Cyclic imide²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ IDNO:39), H-Asn-(Glu)₅-des Pro³⁶,Pro³⁷,Pro³⁸[Cyclicimide²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:40), H-(Lys)₆-desPro³⁶[Trp(O₂)²⁵,Cyclic imide²⁸]Exendin-4(1-39)-Lys₆-NH₂ (SEQ ID NO:48),H-(Lys)₆-des Pro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵,Cyclicimide²⁸]Exendin-4(1-39)-NH₂ (SEQ ID NO:50), H-Asn-(Glu)₅-desPro³,Pro⁷,Pro³⁸[Trp(O₂)²⁵,Cyclic imide²⁸]Exendin-4(1-39)-NH₂ (SEQ IDNO:51), des Pro³⁶,Pro³⁷ Pro³⁸[Trp(O₂)²⁵,Cyclicimide²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:52), H-(Lys)₆-desPro³⁶,Pro³⁷,Pro³⁸[Tip(O₂)²⁵,Cyclic imide²⁸]Exendin-4(1-39)-(Lys)₆-NH₂(SEQ ID NO:53), and H-Asn-(Glu)₅-des Pro³⁶,Pro³⁷,Pro³⁸[Trp(O₂)²⁵, Cyclicimide²⁸]Exendin-4(1-39)-(Lys)₆-NH₂ (SEQ ID NO:54), or a pharmaceuticallyacceptable salt or solvate thereof.
 14. The composition of claim 1,further comprising a pharmaceutically acceptable carrier.
 15. Thecomposition of claim 14, wherein the composition comprises a depotformulation, microspheres, or liposomes or the composition includes astabilized liquid formulation.
 16. A method of treating diabetes type 1or type 2, insulin resistance syndrome, impaired glucose tolerance(IGT), obesity, eating disorders, hyperglycemia, metabolic disorders,and gastric disease, disease states associated with elevated bloodglucose levels, regulation of blood glucose levels, regulation ofgastric emptying, stimulating insulin release, the method comprisingadministering a therapeutically effective amount of the composition ofclaim 1.